**2. Physiological and molecular drought stress responses in sugarcane**

Water stress is one of the most critical environmental abiotic stresses that affect plant's growth and productivity. It was estimated by the International Water Management Institute that by the year 2025, one third of the world will be occupied with severe water scarcity. Moreover, the climate change will induce competition between the use for human consumption and irrigation, which in turn affects the displacement of agriculture to non-irrigated marginal area that reduced in agricultural productivity. When subjected to water deficit or drought stress, plants undergo alteration in physiological started with reduction in protein synthesis, stomatal conductance and photosynthetic rate. Depending on the plants species, drought stress condition will accumulate the compatible solutes to protect cell from serious damage in drought stress tolerant plants. Under rehydration after mild water deficit, almost every plant can return to normal growth, but if the stress was severe, some plants will not survive and dry.

Sugarcane is photosynthetically classified as C4 plant that adapted well in tropical climate. The C4 plants are often considered to be a better adapted to water limitation environments than most other crops, particularly as they are able to maintain leaf photosynthesis with slightly stomatal close and increase in water-use efficiency. The C4 photosynthesis is characterized by the presence of phosphoenolpyruvate carboxylase (PEPC) as the primary carboxylation enzyme located in mesophyll cell, and by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as the secondary carboxylation enzyme located in bundle sheath cells of anatomy C4 leaf. Fixation of CO<sup>2</sup> from atmosphere is catalyzed by PEPC in the mesophyll cells to form C4 acid (malate and/or aspartate) which is then transported into the bundle sheath cells. The metabolites transport process generates a much higher concentration of CO<sup>2</sup> in the bundle sheath cells than the external atmospheres. This elevated concentration of CO<sup>2</sup> at the carboxylation site of Rubisco results in suppression of photorespiration. It was believed that PEPC has high affinity to assimilate CO<sup>2</sup> from atmosphere [11, 12] and allow high rates of photosynthetic carbon assimilation to occur when stomata are slightly closed to prevent evapotranspiration. This PEPC has a significant role in C4 photosynthesis carbon assimilation and is regulated by environmental conditions such as light [13], water stress [14], and nitrogen availability [15]. Although limited reports, the enzymes involved in CO<sup>2</sup> concentration mechanism in C4 plant are considered to play significant role in water resistant [16, 17].

Sugarcane is an important crop to produce sugar and dry condition is commonly observed in sugarcane farm in tropical agriculture. Dry season or drying prior to harvest in irrigated sugarcane cultivation is an important strategy to enhance sucrose content in stem [18, 19]. Gradual water deficit during sugarcane maturation reduce stem elongation and leaf development, but more sucrose become available for storage in stem [18]. In the pathway of sucrose biosynthesis, sucrose-phosphate synthase (SPS) is believed to be a key enzyme for sucrose synthesis in plants [20]. A comparison study on the sucrose content in sugarcane cultivated in different agro-climate showed that dry-land cultivated sugarcane accumulated more sugar compared with wet-land and observation in *Saccharum* species showed that sucrose contents are fluctuated according to the SPS activities [21]. Further physiological study on drought stress revealed that stop watering increased sucrose-phosphate synthase (SPS) along with sucrose accumulation in sugarcane leaf (**Figure 1A**). Similar results were reported that water stress resulted in a stimulation of sucrose synthesis by activation of sucrose-phosphate synthase in spinach [22] and wheat [23]. Addition of ABA increased the SPS activities but did not increase other proteins levels (**Figure 1B**) since the hormone is involved in the signal transduction of gene expression conferring the adaptation [7, 8]. Identification of amino acid residue serine that is responsible for water-stress regulation by phosphorylation mechanism clearly showed that the amino acid is conserved in sugarcane SPS [24]. This experimental result suggests that drought stress induce sucrose accumulation in sugarcane as a mechanism helping the plant adapted to drought conditions. Moreover, overexpression of the gene for SPS has been reported to enhance SPS activity as well as sucrose accumulation in transgenic tomato [25]. The increasing of sucrose accumulation due to overexpression of the gene for SPS enhanced drought stress-tolerance will be an important study to be conducted in sugarcane.

and regulation of signal transduction [4]. Molecular study on the responses of sugarcane to drought stress found the presence of a drought inducible protein named SoDIP22 in the water stress tolerant phenotype of sugarcane [10]. A computer search of protein databases revealed that the sequence of the drought inducible protein exhibited significant similarity to that of members of the ABA stress and ripening-inducible (Asr) protein family, such as 73% identical to rice OsAsr1 protein. The expression of the drought-inducible from sugarcane SoDip22 protein was induced by drought stress and osmotic stress at −0.9 Mpa generated with PEG 6000 and 0.6 M mannitol. The expression of SoDip22 was controlled by the signal transduction pathway through ABA, since exogenous addition of ABA induced the SoDip22 expression, but not other growth regulators. Although the molecular size of SoDip22 was a small protein, only 22 kDa, and has similarity with Asr protein found in the nucleosome fraction which is predicted as a transcription factor, observation of transient expression of the SoDip22 protein did not support the nuclear localization. Interestingly, the protein was inclusively detected in bundle sheath cell of sugarcane leafs and the protein function is predicted to play an important role in the maintenance of water molecule during water deficit

content measured using HLPC. The figures were provided by Dr. Yudhi Rinanto.

**Figure 1.** Enhancement of SPS levels in sugarcane leaves after drought stress (A) and ABA hormone (B) treatments. Two-months old sugarcane plants grown in green house were treated by either drought stress or ABA hormone. The drought stress was initiated by left sugarcane plants without watering and the SPS activity, SPS protein levels, and sucrose contents were measured at indicated times (A). (B) The fully developed youngest leafs were sprayed with ABA solution at indicated concentration for 1 and 2 days. Total proteins were extracted from the fully developed youngest leaves and the SPS activity was measured according to the method described in [21]. The levels of SPS, Rubisco-LSU, and GS (glutamine synthetase) proteins were detected by Western Blot analysis with specific polyclonal antibody against the proteins. The sucrose was extracted from the leaves using mixture of methanol-chloroform-water, and the sucrose

Biotechnology of Drought-Tolerant Sugarcane http://dx.doi.org/10.5772/intechopen.72436 143

Water deficit causes various changes in biochemical reactions, including the production of a complex variety of secondary metabolites. Water stress induces the accumulation of reactive oxygen species (ROS) in plants which are highly reactive or toxic that causes damage to

in the bundle sheath cell.

Drought stress induces a wide range of physiological and biochemical responses in plants, including alteration in gene expression. The change in gene expression was triggered both by ABA-dependent and ABA-independent regulatory mechanism. Furthermore, identification by microarray analysis had classified two groups of drought-inducible genes in *Arabidopsis*. The first group is genes encoding for proteins with the function in abiotic stress tolerance and the second group is comprised of regulatory protein such as various transcription factors

stomatal close and increase in water-use efficiency. The C4 photosynthesis is characterized by the presence of phosphoenolpyruvate carboxylase (PEPC) as the primary carboxylation enzyme located in mesophyll cell, and by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as the secondary carboxylation enzyme located in bundle sheath cells of anatomy

C4 acid (malate and/or aspartate) which is then transported into the bundle sheath cells. The

ation site of Rubisco results in suppression of photorespiration. It was believed that PEPC has

carbon assimilation to occur when stomata are slightly closed to prevent evapotranspiration. This PEPC has a significant role in C4 photosynthesis carbon assimilation and is regulated by environmental conditions such as light [13], water stress [14], and nitrogen availability [15]. Although limited reports, the enzymes involved in CO<sup>2</sup> concentration mechanism in C4 plant

Sugarcane is an important crop to produce sugar and dry condition is commonly observed in sugarcane farm in tropical agriculture. Dry season or drying prior to harvest in irrigated sugarcane cultivation is an important strategy to enhance sucrose content in stem [18, 19]. Gradual water deficit during sugarcane maturation reduce stem elongation and leaf development, but more sucrose become available for storage in stem [18]. In the pathway of sucrose biosynthesis, sucrose-phosphate synthase (SPS) is believed to be a key enzyme for sucrose synthesis in plants [20]. A comparison study on the sucrose content in sugarcane cultivated in different agro-climate showed that dry-land cultivated sugarcane accumulated more sugar compared with wet-land and observation in *Saccharum* species showed that sucrose contents are fluctuated according to the SPS activities [21]. Further physiological study on drought stress revealed that stop watering increased sucrose-phosphate synthase (SPS) along with sucrose accumulation in sugarcane leaf (**Figure 1A**). Similar results were reported that water stress resulted in a stimulation of sucrose synthesis by activation of sucrose-phosphate synthase in spinach [22] and wheat [23]. Addition of ABA increased the SPS activities but did not increase other proteins levels (**Figure 1B**) since the hormone is involved in the signal transduction of gene expression conferring the adaptation [7, 8]. Identification of amino acid residue serine that is responsible for water-stress regulation by phosphorylation mechanism clearly showed that the amino acid is conserved in sugarcane SPS [24]. This experimental result suggests that drought stress induce sucrose accumulation in sugarcane as a mechanism helping the plant adapted to drought conditions. Moreover, overexpression of the gene for SPS has been reported to enhance SPS activity as well as sucrose accumulation in transgenic tomato [25]. The increasing of sucrose accumulation due to overexpression of the gene for SPS enhanced drought stress-tolerance will be an

Drought stress induces a wide range of physiological and biochemical responses in plants, including alteration in gene expression. The change in gene expression was triggered both by ABA-dependent and ABA-independent regulatory mechanism. Furthermore, identification by microarray analysis had classified two groups of drought-inducible genes in *Arabidopsis*. The first group is genes encoding for proteins with the function in abiotic stress tolerance and the second group is comprised of regulatory protein such as various transcription factors

metabolites transport process generates a much higher concentration of CO<sup>2</sup>

sheath cells than the external atmospheres. This elevated concentration of CO<sup>2</sup>

are considered to play significant role in water resistant [16, 17].

important study to be conducted in sugarcane.

from atmosphere is catalyzed by PEPC in the mesophyll cells to form

from atmosphere [11, 12] and allow high rates of photosynthetic

in the bundle

at the carboxyl-

C4 leaf. Fixation of CO<sup>2</sup>

142 Sugarcane - Technology and Research

high affinity to assimilate CO<sup>2</sup>

**Figure 1.** Enhancement of SPS levels in sugarcane leaves after drought stress (A) and ABA hormone (B) treatments. Two-months old sugarcane plants grown in green house were treated by either drought stress or ABA hormone. The drought stress was initiated by left sugarcane plants without watering and the SPS activity, SPS protein levels, and sucrose contents were measured at indicated times (A). (B) The fully developed youngest leafs were sprayed with ABA solution at indicated concentration for 1 and 2 days. Total proteins were extracted from the fully developed youngest leaves and the SPS activity was measured according to the method described in [21]. The levels of SPS, Rubisco-LSU, and GS (glutamine synthetase) proteins were detected by Western Blot analysis with specific polyclonal antibody against the proteins. The sucrose was extracted from the leaves using mixture of methanol-chloroform-water, and the sucrose content measured using HLPC. The figures were provided by Dr. Yudhi Rinanto.

and regulation of signal transduction [4]. Molecular study on the responses of sugarcane to drought stress found the presence of a drought inducible protein named SoDIP22 in the water stress tolerant phenotype of sugarcane [10]. A computer search of protein databases revealed that the sequence of the drought inducible protein exhibited significant similarity to that of members of the ABA stress and ripening-inducible (Asr) protein family, such as 73% identical to rice OsAsr1 protein. The expression of the drought-inducible from sugarcane SoDip22 protein was induced by drought stress and osmotic stress at −0.9 Mpa generated with PEG 6000 and 0.6 M mannitol. The expression of SoDip22 was controlled by the signal transduction pathway through ABA, since exogenous addition of ABA induced the SoDip22 expression, but not other growth regulators. Although the molecular size of SoDip22 was a small protein, only 22 kDa, and has similarity with Asr protein found in the nucleosome fraction which is predicted as a transcription factor, observation of transient expression of the SoDip22 protein did not support the nuclear localization. Interestingly, the protein was inclusively detected in bundle sheath cell of sugarcane leafs and the protein function is predicted to play an important role in the maintenance of water molecule during water deficit in the bundle sheath cell.

Water deficit causes various changes in biochemical reactions, including the production of a complex variety of secondary metabolites. Water stress induces the accumulation of reactive oxygen species (ROS) in plants which are highly reactive or toxic that causes damage to cellular component such as proteins, lipids, carbohydrate, and DNA. The ROS also controls many processes such as cell cycle and programmed cell death [26]. Exposure of plants to drought condition increases production of ROS such as free radical (O<sup>2</sup> \*, superoxide radicals, OH\* hydroxyl radical, HO2 \* perhydroxy radical) and non-radical forms (H<sup>2</sup> O2 , hydrogen peroxide and O2 , singlet oxygen). To ensure survival under drought stress condition, plants have developed efficient antioxidant machinery that is able to scavenger and detoxify ROS [27]. Plants possess enzymatic and non-enzymatic antioxidant defense system to protect plant cell from oxidative stress by scavenging ROS. The enzymatic activity such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and non-enzymatic antioxidants such as ascorbic acid, reduced glutathione, phenolic, alkaloids, and amino acids can work together to scavenge ROS. Water deficit induced the change activities of SOD, CAT, APX, and GR, according to variety and stress intensity in sugarcane. Drought-tolerant sugarcane exhibited higher CAT and APX activities in the early stage of drought, while the activity of GR was highest at the end of drought stress period compared with drought-sensitive sugarcane [28]. The increase of SOD, CAT and APX under drought stress was further confirmed in most tested sugarcane cultivars [29]. Thus, the activities of ROS scavenging enzymes may be used as a marker of water stress tolerant sugarcane.

Although the role of proline on osmotolerance remains controversial in plants, evaluation of transgenic sugarcane overexpressing heterologous *P5CS* gene indicated that proline content significantly increases after 9 days without watering. However, the increasing proline content has no effect on the osmotic adjustment, and the proline may protect sugarcane against the oxidative stress caused by water deficit. The results suggested that proline accumulation acts as a component of antioxidative defense system rather than as an osmotic adjustment [33].

Biotechnology of Drought-Tolerant Sugarcane http://dx.doi.org/10.5772/intechopen.72436 145

The glycine betaine (GB) is one of the compatible solutes and an amphoteric quaternary amine that is considered as the most compatible solute that plays an important role in protecting plants under environmental stress [5, 34]. The GB is synthesized by plants at various capacity, such as spinach and barley accumulate high of GB, whereas *Arabidopsis* and tobacco do not synthesize GB. The role of GB is not only allowing cell to adjust the osmotic potential in their cytoplasm to maintain an appropriate water content, but also protecting protein from the water stress dissociation [34]. When plant cell is exposed to water stress or salt stress, GB stabilizes the structure of macromolecule, helping in maintaining the integrity and proper function of the membrane. Although the detail of the role of GB has not been firmly established, the objective of this book chapter is directed for developing of biotechnology of

Genetic transformation has a potential role to introduce a new trait in plant cell, including the introduction of new pathway for the biosynthesis of compatible solutes and resulting in transgenic plant with improved tolerance to environmental stress. There are many techniques for introducing a new gene into plant cell such as direct transformation using particle bombardments, micro injection or electroporation and indirect transformation using *Agrobacterium* as the vector. Depending on the plant species, *Agrobacterium*-mediated transformation is well established in dicotyledonous plants and less in monocotyledonous plants. The development of *Agrobacterium*-mediated transformation was successfully established for routine genetic

drought-tolerant sugarcane using production of GB in transgenic sugarcane.

transformation in monocotyledonous plants such as rice, maize, and sugarcane.

**transformation for sugarcane**

**3. Development of an efficient method for** *Agrobacterium***-mediated** 

The genetic improvement of sugarcane cultivars has been achieved to increase sugar productivity with the cultivars created by conventional breeding. The sugarcane (*Saccharum officinarum*) cultivars contributed high sucrose production and the backcrosses of *S. officinarum* resulted in elite cultivars of *S. hybrid* with higher yield, improving ratooning ability and disease resistance. However, these improvise achievements would still enable the sugar industries to face current issues on climate changes as well as higher sugar demand due to human population growth. Sugarcane has a highly complex genome, low fertilization and tiny seed which make traditional breeding highly difficult and laborious to create new sugarcane cultivars. Recent progress on molecular techniques could be used in sugarcane varietal improvement as well as the combination between both traditional and molecular breeding. Beginning with development of cell and tissue culture of sugarcane that has emerged as a valuable tool for various research activity in sugarcane improvement and propagation,

Many plants respond to water deficit stress by accumulating non-enzymatic antioxidants defense system to protect from oxidative damage by ROS. Ascorbic acid is one of antioxidants that prevent or minimize the damage caused by ROS. The ascorbic acid has ability to donate electrons in numerous reactions and protect the cell membranes by scavenging the superoxide radical and hydroxyl radical [27]. Furthermore, glutathione is another important antioxidant that is capable of preventing damage caused by ROS. Glutathione exists either in reduced or in oxidized form and it is a crucial metabolite to perform multiple functions including plant responses modulation under abiotic and biotic stresses [30]. Despite ROS scavenging enzymatic and non-enzymatic antioxidants which had been reported to enhance drought tolerance in several transgenic plants [27], their application for developing drought-tolerant sugarcane is still meager.

The accumulation of non-toxic small molecule metabolites referred as compatible solutes or osmoprotectant such as sugar, proline and betaines help plants to survive under osmotic stress [5, 31]. These metabolites may have a role to protect cell membrane and maintain osmotic potential. Studies at physiological, biochemical, and molecular levels suggest that compatibles solutes perform important function in adjustment of plant against salinity and drought stress. Sugar and sugar alcohols have been accepted as osmoprotectant that provide membrane protection and scavenging ROS. The higher accumulation of sugar, such as trehalose, fructans, sucrose, acts as osmoprotectant under drought stress in plants [32]. Sugarcane can accumulate high content of sucrose in storage parenchyma of stem cell that may create osmotic gradient and act as osmoprotectant. Under water deficit, there was a change in stress-related gene expression and sucrose accumulation, but the mechanism responding to the water stress was different. Some genes expression such as genes encoding for asparagine synthase (AS), biosynthesis of proline (OAT), sugar transports were positively correlated, but the genes encoding for proline biosynthetic pathway (P5CS) and the bZIP transcription factor TF1 were negatively correlated with sucrose contents in sugarcane mature culm. The proline content was increased under water deficit condition, but was negatively correlated with sucrose concentration and suggested that proline has no osmoprotectant role in sugarcane [9]. Although the role of proline on osmotolerance remains controversial in plants, evaluation of transgenic sugarcane overexpressing heterologous *P5CS* gene indicated that proline content significantly increases after 9 days without watering. However, the increasing proline content has no effect on the osmotic adjustment, and the proline may protect sugarcane against the oxidative stress caused by water deficit. The results suggested that proline accumulation acts as a component of antioxidative defense system rather than as an osmotic adjustment [33].

cellular component such as proteins, lipids, carbohydrate, and DNA. The ROS also controls many processes such as cell cycle and programmed cell death [26]. Exposure of plants to

plants have developed efficient antioxidant machinery that is able to scavenger and detoxify ROS [27]. Plants possess enzymatic and non-enzymatic antioxidant defense system to protect plant cell from oxidative stress by scavenging ROS. The enzymatic activity such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and non-enzymatic antioxidants such as ascorbic acid, reduced glutathione, phenolic, alkaloids, and amino acids can work together to scavenge ROS. Water deficit induced the change activities of SOD, CAT, APX, and GR, according to variety and stress intensity in sugarcane. Drought-tolerant sugarcane exhibited higher CAT and APX activities in the early stage of drought, while the activity of GR was highest at the end of drought stress period compared with drought-sensitive sugarcane [28]. The increase of SOD, CAT and APX under drought stress was further confirmed in most tested sugarcane cultivars [29]. Thus, the activities of

ROS scavenging enzymes may be used as a marker of water stress tolerant sugarcane.

[27], their application for developing drought-tolerant sugarcane is still meager.

Many plants respond to water deficit stress by accumulating non-enzymatic antioxidants defense system to protect from oxidative damage by ROS. Ascorbic acid is one of antioxidants that prevent or minimize the damage caused by ROS. The ascorbic acid has ability to donate electrons in numerous reactions and protect the cell membranes by scavenging the superoxide radical and hydroxyl radical [27]. Furthermore, glutathione is another important antioxidant that is capable of preventing damage caused by ROS. Glutathione exists either in reduced or in oxidized form and it is a crucial metabolite to perform multiple functions including plant responses modulation under abiotic and biotic stresses [30]. Despite ROS scavenging enzymatic and non-enzymatic antioxidants which had been reported to enhance drought tolerance in several transgenic plants

The accumulation of non-toxic small molecule metabolites referred as compatible solutes or osmoprotectant such as sugar, proline and betaines help plants to survive under osmotic stress [5, 31]. These metabolites may have a role to protect cell membrane and maintain osmotic potential. Studies at physiological, biochemical, and molecular levels suggest that compatibles solutes perform important function in adjustment of plant against salinity and drought stress. Sugar and sugar alcohols have been accepted as osmoprotectant that provide membrane protection and scavenging ROS. The higher accumulation of sugar, such as trehalose, fructans, sucrose, acts as osmoprotectant under drought stress in plants [32]. Sugarcane can accumulate high content of sucrose in storage parenchyma of stem cell that may create osmotic gradient and act as osmoprotectant. Under water deficit, there was a change in stress-related gene expression and sucrose accumulation, but the mechanism responding to the water stress was different. Some genes expression such as genes encoding for asparagine synthase (AS), biosynthesis of proline (OAT), sugar transports were positively correlated, but the genes encoding for proline biosynthetic pathway (P5CS) and the bZIP transcription factor TF1 were negatively correlated with sucrose contents in sugarcane mature culm. The proline content was increased under water deficit condition, but was negatively correlated with sucrose concentration and suggested that proline has no osmoprotectant role in sugarcane [9].

\* perhydroxy radical) and non-radical forms (H<sup>2</sup>

, singlet oxygen). To ensure survival under drought stress condition,

\*, superoxide radi-

O2

, hydro-

drought condition increases production of ROS such as free radical (O<sup>2</sup>

cals, OH\* hydroxyl radical, HO2

gen peroxide and O2

144 Sugarcane - Technology and Research

The glycine betaine (GB) is one of the compatible solutes and an amphoteric quaternary amine that is considered as the most compatible solute that plays an important role in protecting plants under environmental stress [5, 34]. The GB is synthesized by plants at various capacity, such as spinach and barley accumulate high of GB, whereas *Arabidopsis* and tobacco do not synthesize GB. The role of GB is not only allowing cell to adjust the osmotic potential in their cytoplasm to maintain an appropriate water content, but also protecting protein from the water stress dissociation [34]. When plant cell is exposed to water stress or salt stress, GB stabilizes the structure of macromolecule, helping in maintaining the integrity and proper function of the membrane. Although the detail of the role of GB has not been firmly established, the objective of this book chapter is directed for developing of biotechnology of drought-tolerant sugarcane using production of GB in transgenic sugarcane.

Genetic transformation has a potential role to introduce a new trait in plant cell, including the introduction of new pathway for the biosynthesis of compatible solutes and resulting in transgenic plant with improved tolerance to environmental stress. There are many techniques for introducing a new gene into plant cell such as direct transformation using particle bombardments, micro injection or electroporation and indirect transformation using *Agrobacterium* as the vector. Depending on the plant species, *Agrobacterium*-mediated transformation is well established in dicotyledonous plants and less in monocotyledonous plants. The development of *Agrobacterium*-mediated transformation was successfully established for routine genetic transformation in monocotyledonous plants such as rice, maize, and sugarcane.
